Acute myelogenous leukemia (AML) currently kills the majority of afflicted patients despite treatment with combination chemotherapy and allogeneic stem cell transplantation (SCT). Radiolabeled anti-CD45 monoclonal antibodies (Ab) have been shown to improve outcomes for AML in the setting of SCT, but toxicity remains high and cure rates are suboptimal. The objective of this research proposal is to maximize the cure rate of AML using a novel approach employing pretargeted radioimmunotherapy (PRIT) with genetically engineered bispecific antibodies. In Aim 1, we will engineer, express, and purify anti-CD45 x anti-ligand bispecific Abs (scFv2-iFabs) that bind covalently and irreversibly to radiometal ligands. In Aim 2, we will assess the binding characteristics of anti-CD45 x anti-ligand bispecific Abs (scFv2-iFabs) for CD45 antigen and for radiometal ligand chelates in vitro. In Aim 3, we will compare and contrast the pharmacokinetics and biodistributions of pretargeted radioimmunotherapy using the novel molecularly engineered anti-CD45 x anti- ligand bispecific Abs (scFv2-iFabs) with our current gold standard method of PRIT using a streptavidin-biotin pretargeting method. Comparisons will be made in both an AML xenograft model and in a disseminated model of AML. In Aim 4, we will compare and contrast the therapeutic efficacy of pretargeted RIT using the streptavidin-biotin approach with the novel bispecific anti-CD45 x anti-ligand bispecific antibody approach in both xenograft and disseminated AML models. In Aim 5, we will investigate the toxicity and efficacy of combination therapy using anti-CD45 bispecific Ab pretargeting, with and without cytotoxic chemotherapy, in a disseminated model of human AML in SCID mice. We hypothesize that the novel bispecific pretargeted RIT strategy defined in this proposal will amplify the amount of radiation delivered to AML cells, decrease the radiation delivered to the liver, lungs, and other normal organs, improve remission and cure rates, prolong survival, and markedly attenuate toxicities compared to conventional RIT and to streptavidin-biotin PRIT. We anticipate rapid translation of the results of these preclinical experiments into our clinical RIT program for AML.